Bacillus thuringiensis cry gene and protein

ABSTRACT

Compositions and methods for protecting a plant from an insect pest are provided. In particular, novel polynucleotides and the pesticidal polypeptides they encode are provided. Methods of using the novel polynucleotides and pesticidal polypeptides of the invention to protect a plant from an insect pest are further provided. Particular embodiments of the invention provide pesticidal compositions and formulations, DNA constructs, and transformed plants, plant cells, and seeds.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 11/404,297 filed Apr.14, 2006.

FIELD OF THE INVENTION

The present invention relates to the fields of plant molecular biologyand plant pest control.

BACKGROUND OF THE INVENTION

Insect pests are a major factor in the loss of the world's agriculturalcrops. For example, corn rootworm feeding damage or boll weevil damagecan be economically devastating to agricultural producers. Insectpest-related crop loss from corn rootworm alone has reached one billiondollars a year.

Traditionally, the primary methods for impacting insect pestpopulations, such as corn rootworm populations, are crop rotation andthe application of broad-spectrum synthetic chemical pesticides.However, consumers and government regulators alike are becomingincreasingly concerned with the environmental hazards associated withthe production and use of synthetic chemical pesticides. Because of suchconcerns, regulators have banned or limited the use of some of the morehazardous pesticides. Thus, there is substantial interest in developingalternative pesticides.

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria, or another species of insectaffords an environmentally friendly and commercially attractivealternative. Generally speaking, the use of biopesticides presents alower risk of pollution and environmental hazards, and provides agreater target specificity than is characteristic of traditionalbroad-spectrum chemical insecticides. In addition, biopesticides oftencost less to produce and thus improve economic yield for a wide varietyof crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a broad range of insect pestsincluding Lepidoptera, Diptera, Coleoptera, Hemiptera, and others.Bacillus thuringiensis (Bt) and Bacillus popilliae are among the mostsuccessful biocontrol agents discovered to date. Insect pathogenicityhas been attributed to strains of: B. larvae, B. lentimorbus, B.popilliae, B. sphaericus, Bt (Harwook, ed. (1989) Bacillus (PlenumPress), p. 306) and B. cereus (WO 96/10083). Pesticidal activity appearsto be concentrated in parasporal crystalline protein inclusions,although pesticidal proteins have also been isolated from the vegetativegrowth stage of Bacillus. Several genes encoding these pesticidalproteins have been isolated and characterized (see, for example, U.S.Pat. Nos. 5,366,892 and 5,840,868).

Microbial pesticides, particularly those obtained from Bacillus strains,have played an important role in agriculture as alternatives to chemicalpest control. Pesticidal proteins isolated from strains of Bt, known asδ-endotoxins or Cry toxins, are initially produced in an inactiveprotoxin form. These protoxins are proteolytically converted into anactive toxin through the action of proteases in the insect gut. See,Rukmini et al. (2000) Biochimie 82:109-116; Oppert (1999) Arch. InsectBiochem. Phys. 42:1-12; and Carroll et al. (1997) J. InvertebratePathology 70:41-49. Proteolytic activation of the toxin can include theremoval of the N- and C-terminal peptides from the protein, as well asinternal cleavage of the protein. Once activated, the Cry toxin bindswith high affinity to receptors on epithelial cells in the insect gut,thereby creating leakage channels in the cell membrane, lysis of theinsect gut, and subsequent insect death through starvation andsepticemia. See, e.g., Li et al. (1991) Nature 353:815-821.

Recently, agricultural scientists have developed crop plants withenhanced insect resistance by genetically engineering crop plants withpesticidal genes to produce pesticidal proteins from Bacillus. Forexample, corn and cotton plants genetically engineered to produce Crytoxins (see, e.g., Aronson (2002) Cell Mol. Life Sci. 59(3):417-425;Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62(3):775-806) are nowwidely used in American agriculture and have provided the farmer with anenvironmentally friendly alternative to traditional insect-controlmethods. In addition, potatoes genetically engineered to containpesticidal Cry toxins have been developed. These successes with geneticengineering have led researchers to search for novel pesticidal genes,particularly Cry genes. Therefore, novel homologues of known pesticidalgenes are needed.

SUMMARY OF THE INVENTION

Compositions and methods for protecting a plant from a plant pest,particularly an insect pest, are provided. The compositions includenovel nucleic acid molecules, and variants and fragments thereof, thatencode pesticidal polypeptides. The amino acid sequences for the novelpesticidal polypeptides encoded by the nucleotide sequences of theembodiments are further provided. Compositions also include DNAconstructs comprising a promoter operably linked to a nucleotidesequence that encodes a pesticidal polypeptide of the embodiments.Transformed plants, plant cells, seeds, and microorganisms comprising apolynucleotide of the embodiments are further provided.

The novel nucleic acid compositions of the embodiments find use inmethods directed to protecting a plant from an insect pest. The methodscomprise introducing into a plant a polynucleotide construct comprisinga nucleotide sequence that encodes a pesticidal polypeptide of theembodiments operably linked to a promoter that drives expression in aplant. As a result, the pesticidal polypeptide is expressed in the plantand the insect pest is exposed to the protein at the site of insectattack. The presence of the pesticidal polypeptide protects the plantfrom the insect pest.

The embodiments further provide pesticidal compositions and formulationsand methods for their use in controlling insect pests. Pesticidalcompositions comprise a pesticidal polypeptide or transformedmicroorganism comprising a nucleotide sequence encoding a pesticidalpolypeptide of the embodiments. Methods of using these compositions toimpact an insect pest of a plant comprise applying the pesticidalcomposition to the environment of the insect pest.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions directed to protecting a plant from an insectpest are provided. Compositions of the embodiments include novelnucleotide and amino acid sequences for pesticidal polypeptides.Specifically, the embodiments provide isolated nucleic acid molecules,and variants and fragments thereof, comprising the nucleotide sequenceset forth in SEQ ID NO:1. Pesticidal polypeptides encoded by the novelnucleic acids of the embodiments are also provided. More particularly,compositions include pesticidal polypeptides having the amino acidsequence set forth in SEQ ID NO: 2, and variants and fragments thereof.Plants, plant cells, seeds, microorganisms, and DNA constructscomprising a nucleotide sequence of the embodiments that encodes apesticidal polypeptide are also disclosed herein. Pesticidalcompositions comprising an isolated pesticidal polypeptide of theembodiments, or a microorganism that expresses a nucleic acid of theembodiments, in combination with a carrier are further provided. Thecompositions of the embodiments find use in methods for protecting aplant from an insect pest or for impacting an insect pest.

The nucleic acid molecules of the embodiments comprise nucleotidesequences that are homologous to known pesticidal genes, particularly BtCry genes, more particularly Cry8A and Cry8B genes. The predicted aminoacid sequence encoded by a nucleotide sequence of the embodiments isalso disclosed as SEQ ID NO: 2. The present compositions can be used topractice the methods of the embodiments.

Methods directed to protecting a plant from a plant pest, particularlyan insect pest, are provided. By “protecting a plant from an insectpest,” limiting or eliminating insect pest-related damage to a plant by,for example, inhibiting the ability of the insect pest to grow, feed,and/or reproduce or by killing the insect pest is intended. The methodscomprise introducing into a plant a polynucleotide construct comprisinga nucleotide sequence that encodes a pesticidal polypeptide of theembodiments operably linked to a promoter that drives expression in aplant. As a result, the pesticidal polypeptide is expressed in the plantand the insect pest is exposed to the protein at the site of insectattack. The presence of the pesticidal polypeptide protects the plantfrom the insect pest.

While the embodiments do not depend on a particular biological mechanismfor protecting a plant from an insect pest, expression of the nucleotidesequences of the embodiments in a plant can result in the production ofactive pesticidal polypeptides that increase the resistance of the plantto insect pests. The transgenic plants of the embodiments find use inagriculture in methods for protecting plants from insect pests and forimpacting insect pests. Certain embodiments of the invention providetransformed crop plants, such as, for example, potato plants, which finduse in methods for impacting the Colorado potato beetle.

In other embodiments, the pesticidal polypeptides encoded by thepolynucleotides of the embodiments are disclosed as well as methods forusing these polypeptides. Compositions and formulations comprising apesticidal polypeptide, or variant or fragment thereof, are useful inmethods for impacting an insect pest. “Impact an insect pest” or“impacting an insect pest” is intended to mean, for example, deterringthe insect pest from feeding further on the plant, harming the insectpest, or killing the insect pest. In this manner, the embodimentsfurther provide a method for impacting an insect pest of a plantcomprising applying, for example, a composition or formulationcomprising a pesticidal polypeptide to the environment of the insectpest. In one embodiment, the pesticidal polypeptide is combined with acarrier for subsequent application to the environment of the insectpest. While the embodiments are not bound by any theory of operation, inone embodiment, an insect pest ingests the pesticidal polypeptide,thereby impacting the insect pest.

One of skill in the art would recognize that the compositions andmethods of the embodiments can be used alone or in combination withother compositions and methods for controlling insect pests that impactplants. For example, the embodiments may be used in conjunction withother pesticidal proteins or traditional chemical pesticides.

“Pesticidal gene” or “pesticidal polynucleotide” refers to a nucleotidesequence that encodes a polypeptide that exhibits pesticidal activity.As used herein, the term “pesticidal activity” refers to the ability ofa substance, such as a polypeptide, to inhibit the growth, feeding, orreproduction of an insect pest and/or to kill the insect pest. A“pesticidal polypeptide,” “pesticidal protein,” or “insect toxin” isintended to mean a protein having pesticidal activity.

As used herein, the terms “pesticidal activity” and “insecticidalactivity” are used synonymously to refer to activity of an organism or asubstance (such as, for example, a protein) that can be measured by, butis not limited to, pest mortality, pest weight loss, pest repellency,and other behavioral and physical changes of a pest after feeding andexposure for an appropriate length of time. In this manner, pesticidalactivity impacts at least one measurable parameter of pest fitness.Assays for assessing pesticidal activity are well known in the art. See,e.g., U.S. Pat. Nos. 6,570,005 and 6,339,144.

The preferred developmental stage for testing for pesticidal activity islarvae or immature forms of these above-mentioned insect pests. Theinsects may be reared in total darkness at from about 20° C. to about30° C. and from about 30% to about 70% relative humidity. Bioassays maybe performed as described in Czapla and Lang (1990) J. Econ. Entomol.83(6):2480-2485. Methods of rearing insect larvae and performingbioassays are well known to one of ordinary skill in the art.

A wide variety of bioassay techniques for assessing pesticidal activityis known to one skilled in the art. General procedures include additionof the experimental compound or organism to the diet source in anenclosed container. Pesticidal activity can be measured by, but is notlimited to, changes in mortality, weight loss, attraction, repellencyand other behavioral and physical changes after feeding and exposure foran appropriate length of time.

In some embodiments of the invention, the pesticidal gene encodes aBacillus thuringiensis (Bt) toxin, particularly a homologue of a knownCry toxin. “Bt” or “Bacillus thuringiensis” toxin is intended to meanthe broader class of toxins found in various strains of Bt, whichincludes such toxins as, for example, the vegetative insecticidalproteins and the δ-endotoxins. See, for example, Crickmore et al. (1998)Microbiol. Molec. Biol. Rev. 62:807-813; and Crickmore et al. (2004)Bacillus Thuringiensis Toxin Nomenclature atlifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt, both of which are hereinincorporated by reference in their entirety. The vegetative insecticidalproteins (for example, members of the VIP1, VIP2, or VIP3 classes) aresecreted insecticidal proteins that undergo proteolytic processing bymidgut insect fluids. They have pesticidal activity against a broadspectrum of Lepidopteran insects. See, for example, U.S. Pat. No.5,877,012, herein incorporated by reference in its entirety. The Btδ-endotoxins are toxic to larvae of a number of insect pests, includingmembers of the Lepidoptera, Diptera, and Coleoptera orders. These insecttoxins include, but are not limited to, the Cry toxins, including, forexample, Cry1, Cry3, Cry5, Cry8, and Cry9. Of particular interest arepesticidal genes that are homologous to known Cry8 genes.

The Bt toxins are a family of insecticidal proteins that are synthesizedas protoxins and crystallize as parasporal inclusions. When ingested byan insect pest, the microcrystal structure is dissolved by the alkalinepH of the insect midgut, and the protoxin is cleaved by insect gutproteases to generate the active toxin. The activated Bt toxin binds toreceptors in the gut epithelium of the insect, causing membrane lesionsand associated swelling and lysis of the insect gut. Insect deathresults from starvation and septicemia. See, e.g., Li et al. (1991)Nature 353:815-821.

The protoxin form of the Cry toxins contains a crystalline formingsegment. A comparison of the amino acid sequences of active Cry toxinsof different specificities further reveals five highly-conservedsequence blocks. Structurally, the Cry toxins comprise three distinctdomains, which are, from the N- to C-terminus: a cluster of sevenalpha-helices implicated in pore formation (referred to as “domain 1”),three anti-parallel beta sheets implicated in cell binding (referred toas “domain 2”), and a beta sandwich (referred to as “domain 3”). Thelocation and properties of these domains are known to those of skill inthe art. See, for example, Li et al. (1991) supra and Morse et al.(2001) Structure 9:409-417.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

The use of the term “polynucleotide” or “nucleotide” is not intended tolimit the embodiments to polynucleotides comprising DNA. Those ofordinary skill in the art will recognize that polynucleotides, cancomprise ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the embodiments also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogues of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

Polypeptides of the embodiments can be produced either from a nucleicacid disclosed herein, or by the use of standard molecular biologytechniques. For example, a truncated protein of the embodiments can beproduced by expression of a recombinant nucleic acid of the embodimentsin an appropriate host cell, or alternatively by a combination of exvivo procedures, such as protease digestion and purification.

The embodiments encompass isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the embodiments or biologically active portionthereof is recombinantly produced, optimally culture medium representsless than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed polynucleotides and proteinsencoded thereby are also encompassed by the embodiments. The term“fragment” is intended to mean a portion of the polynucleotide or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a polynucleotide may encode protein fragments that retainthe biological activity of the native protein and hence have pesticidalactivity. Alternatively, fragments of a polynucleotide that are usefulas hybridization probes generally do not encode fragment proteinsretaining biological activity. Thus, fragments of a nucleotide sequencemay range from at least about 20 nucleotides, about 50 nucleotides,about 100 nucleotides, and up to the full-length polynucleotidesencoding the proteins of the embodiments.

A fragment of a pesticidal polynucleotide that encodes a biologicallyactive portion of a pesticidal protein of the embodiments will encode atleast 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, or 650 contiguous amino acids, or up to the total number of aminoacids present in a full-length pesticidal protein of the embodiments(for example, 719 amino acids for SEQ ID NO:2). Fragments of apesticidal polynucleotide that are useful as hybridization probes or PCRprimers generally need not encode a biologically active portion of apesticidal protein.

Thus, a fragment of a pesticidal polynucleotide may encode abiologically active portion of a pesticidal polypeptide, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a pesticidalpolypeptide can be prepared by isolating a portion of one of thepesticidal polynucleotides of the embodiments, expressing the encodedportion of the pesticidal protein (e.g., by recombinant expression invitro), and assessing the activity of the encoded portion of thepesticidal protein. Polynucleotides that are fragments of a pesticidalgene comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,1,400, 1,450, 1,500, 1,550, 1,600, 1,650, 1,700, 1,750, 1,800, 1,850,1,900, 1,950, or 2,000 contiguous nucleotides, or up to the number ofnucleotides present in a full-length pesticidal polynucleotide disclosedherein (for example, 2166 nucleotides for SEQ ID NO:1).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of one ofthe pesticidal polypeptides of the embodiments. Naturally occurringallelic variants such as these can be identified with the use ofwell-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotide, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a pesticidal protein ofthe embodiments. Generally, variants of a particular polynucleotide ofthe embodiments will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to that particular polynucleotide as determinedby sequence alignment programs and parameters described elsewhereherein.

Variants of a particular polynucleotide of the embodiments (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 2 is disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described elsewhere herein. Where any given pairof polynucleotides of the embodiments is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the embodiments are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, pesticidal activity as described herein. Such variantsmay result from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a native pesticidalpolypeptide of the embodiments will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to the amino acid sequence forthe native protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the embodiments may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

The proteins of the embodiments may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the pesticidalproteins can be prepared by mutations in the DNA. Methods formutagenesis and polynucleotide alterations are well known in the art.See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.4,873,192; Walker and Gaastra, eds. (1983) Techniques in MolecularBiology (MacMillan Publishing Company, New York) and the referencescited therein. Guidance as to appropriate amino acid substitutions thatdo not affect biological activity of the protein of interest may befound in the model of Dayhoff et al. (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal

Thus, the genes and polynucleotides of the embodiments include both thenaturally occurring sequences as well as mutant forms. Likewise, theproteins of the embodiments encompass both naturally occurring proteinsas well as variations and modified forms thereof. Such variants willcontinue to possess the desired pesticidal activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity of apesticidal polypeptide can be evaluated by, for example, insect-feedingassays. See, e.g., Marrone et al. (1985) J. Econ. Entomol. 78:290-293and Czapla and Lang (1990) supra, herein incorporated by reference.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different pesticidalpolypeptide coding sequences can be manipulated to create a newpesticidal polypeptide possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled betweenthe pesticidal gene of the embodiments and other known pesticidal genesto obtain a new gene coding for a protein with an improved property ofinterest, such as an increased pesticidal activity. Strategies for suchDNA shuffling are known in the art. See, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore etal. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291;and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The polynucleotides of the embodiments can be used to isolatecorresponding sequences from other organisms, particularly othermicroorganisms. In this manner, methods such as PCR, hybridization, andthe like can be used to identify such sequences based on their sequencehomology to the sequences set forth herein. Sequences isolated based ontheir sequence identity to the entire pesticidal sequences set forthherein or to variants and fragments thereof are encompassed by theembodiments. Such sequences include sequences that are orthologs of thedisclosed sequences. “Orthologs” is intended to mean genes derived froma common ancestral gene and which are found in different species as aresult of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions oforthologs are often highly conserved among species. Thus, isolatedpolynucleotides that encode for a pesticidal polypeptide and whichhybridize under stringent conditions to the pesticidal sequencesdisclosed herein, or to variants or fragments thereof, are encompassedby the embodiments.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the pesticidal polynucleotides ofthe embodiments. Methods for preparation of probes for hybridization andfor construction of cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

For example, the entire pesticidal polynucleotide disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding pesticidal polynucleotide andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique amongpesticidal polynucleotide sequences and are optimally at least about 10nucleotides in length, and most optimally at least about 20 nucleotidesin length. Such probes may be used to amplify corresponding pesticidalpolynucleotide from a chosen organism by PCR. This technique may be usedto isolate additional coding sequences from a desired organism or as adiagnostic assay to determine the presence of coding sequences in anorganism. Hybridization techniques include hybridization screening ofplated DNA libraries (either plaques or colonies; see, for example,Sambrook et al. (1989) supra.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the thermal melting point (T_(m))can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)supra.).

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of theembodiments. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the embodiments. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See ncbi.nlm.nih.gov. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The pesticidal polynucleotides of the embodiments can be provided in DNAconstructs (or expression cassettes) for expression in the plant ormicroorganism of interest. The construct will include 5′ and 3′regulatory sequences operably linked to a pesticidal polynucleotide ofthe embodiments. “Operably linked” is intended to mean a functionallinkage between two or more elements. For example, an operable linkagebetween a polynucleotide of interest and a regulatory sequence (i.e., apromoter) is a functional link that allows for expression of thepolynucleotide of interest. Operably linked elements may be contiguousor non-contiguous. When used to refer to the joining of two proteincoding regions, by operably linked is intended that the coding regionsare in the same reading frame. The construct may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multiple DNAconstructs. Such a DNA construct is provided with a plurality ofrestriction sites and/or recombination sites for insertion of apolynucleotide of the embodiments to be under the transcriptionalregulation of the regulatory regions. The DNA construct may additionallycontain selectable marker genes.

The DNA construct will include in the 5′-3′ direction of transcription,a transcriptional initiation region, translational initiation region, aheterologous nucleotide sequence of interest (i.e. a sequence of theembodiments), a translational termination region and, optionally, atranscriptional termination region functional in the host organism. Theregulatory regions (i.e., promoters, transcriptional regulatory regions,and translational termination regions) and/or the polynucleotide of theembodiments may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theembodiments may be heterologous to the host cell or to each other. Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The optionally included termination region may be native with thetranscriptional initiation region, may be native with the operablylinked polynucleotide of interest, may be native with the plant host, ormay be derived from another source (i.e., foreign or heterologous) tothe promoter, the polynucleotide of interest, the host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627-9639. In particular embodiments, the potato proteaseinhibitor II gene (PinII) terminator is used. See, for example, Keil etal. (1986) Nucl. Acids Res. 14:5641-5650; and An et al. (1989) PlantCell 1:115-122, herein incorporated by reference in their entirety.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The DNA constructs may additionally contain 5′ leader sequences. Suchleader sequences can act to enhance translation. Translation leaders areknown in the art and include: picornavirus leaders, for example, EMCVleader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al.(1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968.

In preparing the DNA construct, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The DNA construct can also comprise a selectable marker gene for theselection of transformed cells. Selectable marker genes are utilized forthe selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol. Bioeng. 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan fluorescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol. 129:913-42),and yellow fluorescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph. D. Thesis, University of Heidelberg; Reines et al. (1993) Proc.Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph. D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Hand of Experimental Pharmacology, Vol.78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724.Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

A number of promoters can be used in the practice of the embodiments,including the native promoter of the polynucleotide sequence ofinterest. The promoters can be selected based on the desired outcome.The nucleic acids can be combined with constitutive, tissue-preferred,or other promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Generally, it will be beneficial to express the gene from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819, herein incorporated byreference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the embodiments. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced pesticidalprotein expression within a particular plant tissue, particularly withina tissue that is likely to be the target of pest attack. In particularembodiments, a pesticidal polypeptide is selectively expressed intissues where insect-related damage is likely to occur. Tissue-preferredpromoters include Yamamoto et al. (1997) Plant J. 12(2):255-265;Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al.(1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) TransgenicRes. 6(2):157-168; Rinehart et al. (1996) Plant Physiol.112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535;Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al.(1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. CellDiffer. 20:181-196; Orozco et al. (1993) Plant Mol Biol.23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed roIC and roID root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179. Other root-preferred promoters of interest aredisclosed in U.S. patent application Ser. No. 11/022,111, entitled“Maize Metallothionein Promoter,” filed Dec. 22, 2004, and U.S. patentapplication Ser. No. 11/022,449, entitled “Maize Metallothionein 2Promoter and Methods of Use,” filed Dec. 22, 2004, both of which areherein incorporated by reference in their entirety.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); mi1ps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is an endosperm-specificpromoter. Globulin 1 (Glb-1) is a representative embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, etc. See also WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference.

Where low level expression is desired, weak promoters will be used.Generally, by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By low level is intendedat levels of about 1/1000 transcripts to about 1/100,000 transcripts toabout 1/500,000 transcripts. Alternatively, it is recognized that weakpromoters also encompasses promoters that are expressed in only a fewcells and not in others to give a total low level of expression. Where apromoter is expressed at unacceptably high levels, portions of thepromoter sequence can be deleted or modified to decrease expressionlevels.

Such weak constitutive promoters include, for example, the core promoterof the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also,U.S. Pat. No. 6,177,611, herein incorporated by reference.

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a sequence into a plant, only that thepolynucleotide or polypeptides gains access to the interior of at leastone cell of the plant. Methods for introducing polynucleotide orpolypeptides into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and,5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize);Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-VanSlogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the nucleotide sequences of the embodiments canbe provided to a plant using a variety of transient transformationmethods. Such transient transformation methods include, but are notlimited to, the introduction of the pesticidal protein or variants andfragments thereof directly into the plant or the introduction of thepesticidal polypeptide transcript into the plant. Such methods include,for example, microinjection or particle bombardment. See, for example,Crossway et al. (1986) Mol. Gen. Genet. 202:179-185; Nomura et al.(1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci.USA 91:2176-2180 and Hush et al. (1994) J. Cell Science 107:775-784, allof which are herein incorporated by reference. Alternatively, thepesticidal polynucleotide can be transiently transformed into the plantusing techniques known in the art. Such techniques include viral vectorsystem and the precipitation of the polynucleotide in a manner thatprecludes subsequent release of the DNA. Thus, the transcription fromthe particle-bound DNA can occur, but the frequency with which itsreleased to become integrated into the genome is greatly reduced. Suchmethods include the use particles coated with polyethyleneimine (PEI;Sigma #P3143).

In other embodiments, the polynucleotide of the embodiments may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the embodiments within a viral DNA or RNAmolecule. It is recognized that the a pesticidal polypeptide of theembodiments may be initially synthesized as part of a viral polyprotein,which later may be processed by proteolysis in vivo or in vitro toproduce the desired recombinant protein. Further, it is recognized thatpromoters of the embodiments also encompass promoters utilized fortranscription by viral RNA polymerases. Methods for introducingpolynucleotides into plants and expressing a protein encoded therein,involving viral DNA or RNA molecules, are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367,5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221;herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855, and WO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the embodiments can be contained in a transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site that is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the embodiments provide transformed seed (also referredto as “transgenic seed”) having a polynucleotide of the embodiments, forexample, a DNA construct of the embodiments, stably incorporated intotheir genome.

Pedigree breeding starts with the crossing of two genotypes, such as anelite line of interest and one other elite inbred line having one ormore desirable characteristics (i.e., having stably incorporated apolynucleotide of the embodiments, having a modulated activity and/orlevel of the polypeptide of the embodiments, etc.) that complements theelite line of interest. If the two original parents do not provide allthe desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneous lines asa result of self-pollination and selection. Typically in the pedigreemethod of breeding, five or more successive filial generations ofselfing and selection is practiced: F1→F2; F2→F3; F3→F4; F4→F5, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed inbred. In specificembodiments, the inbred line comprises homozygous alleles at about 95%or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify anelite line of interest and a hybrid that is made using the modifiedelite line. As discussed previously, backcrossing can be used totransfer one or more specifically desirable traits from one line, thedonor parent, to an inbred called the recurrent parent, which hasoverall good agronomic characteristics yet lacks that desirable trait ortraits. However, the same procedure can be used to move the progenytoward the genotype of the recurrent parent but at the same time retainmany components of the non-recurrent parent by stopping the backcrossingat an early stage and proceeding with selfing and selection. Forexample, an F1, such as a commercial hybrid, is created. This commercialhybrid may be backcrossed to one of its parent lines to create a BC1 orBC2. Progeny are selfed and selected so that the newly developed inbredhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the non-recurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newhybrids and breeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of a maize inbred line of interest, comprising thesteps of crossing a plant of the maize inbred line of interest with adonor plant comprising a mutant gene or transgene conferring a desiredtrait (i.e., resistance to insect pests), selecting an F1, progeny plantcomprising the mutant gene or transgene conferring the desired trait,and backcrossing the selected F1, progeny plant to the plant of themaize inbred line of interest. This method may further comprise the stepof obtaining a molecular marker profile of the maize inbred line ofinterest and using the molecular marker profile to select for a progenyplant with the desired trait and the molecular marker profile of theinbred line of interest. In the same manner, this method may be used toproduce an F1, hybrid seed by adding a final step of crossing thedesired trait conversion of the maize inbred line of interest with adifferent maize plant to make F1, hybrid maize seed comprising a mutantgene or transgene conferring the desired trait.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross-pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and topcrossing. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross-pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self-pollination, directed-pollination could beused as part of the breeding program.

Mutation breeding is one of many methods that could be used to introducenew traits into an elite line. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development” Fehr (1993)(Macmillan Publishing Company), the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of elite lines that comprisessuch mutations.

In certain embodiments the polynucleotides of the embodiments can bestacked with any combination of polynucleotide sequences of interest inorder to create plants with a desired trait. A trait, as used herein,refers to the phenotype derived from a particular sequence or groups ofsequences. For example, the polynucleotides of the embodiments may bestacked with any other polynucleotides encoding polypeptides havingpesticidal and/or insecticidal activity, such as other Bt toxic proteins(described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109), lectins (Van Damme etal. (1994) Plant Mol. Biol. 24:825, pentin (described in U.S. Pat. No.5,981,722), and the like. The combinations generated can also includemultiple copies of any one of the polynucleotides of interest. Thepolynucleotides of the embodiments can also be stacked with any othergene or combination of genes to produce plants with a variety of desiredtrait combinations including, but not limited to, traits desirable foranimal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);balanced amino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802; and 5,703,409); barley high lysine (Williamson etal. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122) and highmethionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279;Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) PlantMol. Biol. 12:123)); increased digestibility (e.g., modified storageproteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); andthioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3,2001)); the disclosures of which are herein incorporated by reference.

The polynucleotides of the embodiments can also be stacked with traitsdesirable for disease or herbicide resistance (e.g., fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene)); and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils(e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase),starch synthases (SS), starch branching enzymes (SBE), and starchdebranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S.Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs));the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the embodiments with polynucleotidesproviding agronomic traits such as male sterility (e.g., see U.S. Pat.No. 5,583,210), stalk strength, flowering time, or transformationtechnology traits such as cell cycle regulation or gene targeting (e.g.,WO 99/61619, WO 00/17364, and WO 99/25821); the disclosures of which areherein incorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, cross-breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855,and WO 99/25853, all of which are herein incorporated by reference.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plant can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of theembodiments, provided that these parts comprise the introducedpolynucleotides.

The sequences of the embodiments may be used for transformation andprotection of any plant species, including, but not limited to, monocotsand dicots. Examples of plant species of interest include, but are notlimited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica species useful as sources of seedoil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the embodiments include, forexample, pines such as loblolly pine (Pinus taeda), slash pine (Pinuselliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinuscontorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsugamenziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Piceaglauca); redwood (Sequoia sempervirens); true firs such as silver fir(Abies amabilis) and balsam fir (Abies balsamea); and cedars such asWestern red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparisnootkatensis). In specific embodiments, plants included are crop plants(for example, corn, alfalfa, sunflower, Brassica, soybean, cotton,safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In otherembodiments, corn and soybean plants are optimal, and in yet otherembodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mung bean, lima bean, fava bean, lentils,chickpea, etc.

Pesticidal compositions are also encompassed by the embodiments.Pesticidal compositions may comprise pesticidal polypeptides ormicroorganisms comprising a nucleotide sequence that encodes apesticidal polypeptide. The pesticidal compositions of the embodimentsmay be applied to the environment of a plant pest, as described hereinbelow, thereby protecting a plant from a plant pest. Moreover, apesticidal composition can be formulated with an acceptable carrier thatis, for example, a suspension, a solution, an emulsion, a dustingpowder, a dispersible granule, a wettable powder, and an emulsifiableconcentrate, an aerosol, an impregnated granule, an adjuvant, a coatablepaste, and also encapsulations in, for example, polymer substances.

A gene encoding a pesticidal polypeptide of the embodiments,particularly a Bt Cry toxin, may be introduced into any suitablemicrobial host according to standard methods in the art. For example,microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, and to provide for stablemaintenance and expression of the gene expressing the pesticidalprotein.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli andAzotobacter vinlandii, and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

Other illustrative prokaryotes, both Gram-negative and gram-positive,include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium;Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas,Vibrio, Desulfovibrio, and Spirillum; Lactobacillaceae;Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae;and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetesand Ascomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Microbial host organisms of particular interest include yeast, such asRhodotorula spp., Aureobasidium spp., Saccharomyces spp., andSporobolomyces spp., phylloplane organisms such as Pseudomonas spp.,Erwinia spp., and Flavobacterium spp., and other such organisms,including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bt, Escherichia coli, Bacillus subtilis, and the like.

Genes encoding the pesticidal polypeptides of the embodiments can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver pesticidal proteins to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the pesticidal polypeptides of theembodiments can be introduced into a root-colonizing Bacillus cereus bystandard methods known in the art.

Genes encoding pesticidal proteins can be introduced, for example, intothe root-colonizing Bacillus by means of electrotransformation.Specifically, genes encoding the pesticidal proteins can be cloned intoa shuttle vector, for example, pHT3101 (Lerecius et al. (1989) FEMSMicrobiol. Letts. 60:211-218. The shuttle vector pHT3101 containing thecoding sequence for the particular pesticidal gene can, for example, betransformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius et al. (1989) FEMS Microbiol. Letts.60:211-218).

Methods are provided for protecting a plant from a plant pest comprisingapplying an effective amount of a pesticidal protein or composition ofthe embodiments to the environment of the pest. By “effective amount” isintended an amount of a protein or composition sufficient to control aplant pest. The pesticidal proteins and compositions can be applied tothe environment of the pest by methods known to those of ordinary skillin the art.

The pesticidal compositions of the embodiments may be obtained by theaddition of a surface-active agent, an inert carrier, a preservative, ahumectant, a feeding stimulant, an attractant, an encapsulating agent, abinder, an emulsifier, a dye, a UV protective, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaricides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetpathogens. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the embodiments are normally applied in the formof compositions and can be applied to the crop area, plant, or seed tobe treated. For example, the compositions of the embodiments may beapplied to grain in preparation for or during storage in a grain bin orsilo, etc. The compositions of the embodiments may be appliedsimultaneously or in succession with other compounds. Methods ofapplying an active ingredient of the embodiments or an agrochemicalcomposition of the embodiments that contains at least one of thepesticidal proteins, more particularly Cry toxins, of the embodimentsinclude, but are not limited to, foliar application, seed coating, andsoil application. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest orpathogen.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono- or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The pesticidal compositions of the embodiments can be in a suitable formfor direct application or as a concentrate of primary composition thatrequires dilution with a suitable quantity of water or other diluentbefore application. The concentration of the pesticidal polypeptide willvary depending upon the nature of the particular formulation,specifically, whether it is a concentrate or to be used directly. Thecomposition contains 1 to 98% of a solid or liquid inert carrier, and 0to 50%, preferably 0.1 to 50% of a surfactant. These compositions willbe administered at the labeled rate for the commercial product,preferably about 0.01 lb.-5.0 lb. per acre when in dry form and at about0.01 pts.-10 pts. per acre when in liquid form.

In a further embodiment, the compositions, as well as the transformedmicroorganisms and pesticidal proteins, of the embodiments can betreated prior to formulation to prolong the pesticidal activity whenapplied to the environment of a target pest as long as the pretreatmentis not deleterious to the activity. Such treatment can be by chemicaland/or physical means as long as the treatment does not deleteriouslyaffect the properties of the composition(s). Examples of chemicalreagents include but are not limited to halogenating agents; aldehydessuch a formaldehyde and glutaraldehyde; anti-infectives, such aszephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freemanand Co.).

The pesticidal compositions of the embodiments can be applied to theenvironment of a plant pest by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pest has begun to appear orbefore the appearance of pest as a protective measure. For example, thepesticidal protein and/or transformed microorganisms of the embodimentsmay be mixed with grain to protect the grain during storage. It isgenerally important to obtain good control of pest in the early stagesof plant growth, as this is the time when the plant can be most severelydamaged. The compositions of the embodiments can conveniently contain aninsecticide if this is thought necessary. In one embodiment, thecomposition is applied directly to the soil, at a time of planting, ingranular form of a composition of a carrier and dead cells of a Bacillusstrain or transformed microorganism of the embodiments. Anotherembodiment is a granular form of a composition comprising anagrochemical such as, for example, a herbicide, an insecticide, afertilizer, an inert carrier, and dead cells of a Bacillus strain ortransformed microorganism of the embodiments.

Compositions of the embodiments find use in protecting plants, seeds,and plant products in a variety of ways. For example, the compositionscan be used in a method that involves placing an effective amount of thepesticidal composition in the environment of the pest by a procedureselected from the group consisting of spraying, dusting, broadcasting,or seed coating.

Before plant propagation material (fruit, tuber, bulb, corm, grains,seed), but especially seed, is sold as a commercial product, it iscustomarily treated with a protective coating comprising herbicides,insecticides, fungicides, bactericides, nematicides, molluscicides, ormixtures of several of these preparations, if desired together withfurther carriers, surfactants, or application-promoting adjuvantscustomarily employed in the art of formulation to provide protectionagainst damage caused by bacterial, fungal, or animal pests. In order totreat the seed, the protective coating may be applied to the seedseither by impregnating the tubers or grains with a liquid formulation orby coating them with a combined wet or dry formulation. In addition, inspecial cases, other methods of application to plants are possible,e.g., treatment directed at the buds or the fruit.

The plant seed of the embodiments comprising a DNA molecule comprising anucleotide sequence encoding a pesticidal polypeptide of the embodimentsmay be treated with a seed protective coating comprising a seedtreatment compound, such as, for example, captan, carboxin, thiram,methalaxyl, pirimiphos-methyl, and others that are commonly used in seedtreatment. Alternatively, a seed of the embodiments comprises a seedprotective coating comprising a pesticidal composition of theembodiments is used alone or in combination with one of the seedprotective coatings customarily used in seed treatment.

The methods and compositions of the embodiments may be effective againsta variety of pests. For purposes of the embodiments, pests include, butare not limited to, insects, fungi, bacteria, nematodes, acarids,protozoan pathogens, animal-parasitic liver flukes, and the like. Pestsof particular interest are insect pests, particularly insect pests thatcause significant damage to agricultural plants.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery, ornamentals, food andfiber, public and animal health, domestic and commercial structure,household, and stored product pests. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera and Lepidoptera. These include larvae of the orderLepidoptera, such as armyworms, cutworms, loopers, and heliothines inthe family Noctuidae (e.g., fall armyworm (Spodoptera frugiperda J. E.Smith), beet armyworm (Spodoptera exigua Hübner), bertha armyworm(Mamestra configurata Walker), black cutworm (Agrotis ipsilon Hufnagel),cabbage looper (Trichoplusia ni Hübner), soybean looper (Pseudoplusiaincludens Walker), velvetbean caterpillar (Anticarsia gemmatalisHübner), green cloverworm (Hypena scabra Fabricius) tobacco budworm(Heliothis virescens Fabricius), granulate cutworm (Agrotis subterraneaFabricius), armyworm (Pseudaletia unipuncta Haworth) western cutworm(Agrotis orthogonia Morrison)); borers, casebearers, webworms,coneworms, cabbageworms and skeletonizers from the family Pyralidae(e.g., European corn borer (Ostrinia nubilalis Hübner), navel orangeworm(Amyelois transitella Walker), corn root webworm (Crambus caliginosellusClemens), sod webworm (Herpetogramma licarsisalis Walker), sunflowermoth (Homoeosoma electellum Hulst), lesser cornstalk borer (Elasmopalpuslignosellus Zeller)); leafrollers, budworms, seed worms, and fruit wormsin the family Tortricidae (e.g., codling moth (Cydia pomonellaLinnaeus), grape berry moth (Endopiza viteana Clemens), oriental fruitmoth (Grapholita molesta Busck), sunflower bud moth (Suleima helianthanaRiley)); and many other economically important lepidoptera(e.g.,diamondback moth (Plutella xylostella Linnaeus), pink bollworm(Pectinophora gossypiella Saunders), gypsy moth (Lymantria disparLinnaeus)); nymphs and adults of the order Blattodea includingcockroaches from the families Blattellidae and Blattidae (e.g., orientalcockroach (Blatta orientalis Linnaeus), Asian cockroach (Blatellaasahinai Mizukubo), German cockroach (Blattella germanica Linnaeus),brown banded cockroach (Supella longipalpa Fabricius), Americancockroach (Periplaneta americana Linnaeus), brown cockroach (Periplanetabrunnea Burmeister), Madeira cockroach (Leucophaea maderae Fabricius));foliar feeding larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae, and Curculionidae(e.g., boll weevil (Anthonomus grandis Boheman), rice water weevil(Lissorhoptrus oryzophilus Kuschel), granary weevil (Sitophilusgranarius Linnaeus), rice weevil (Sitophilus oryzae Linnaeus), cloverleaf weevil (Hypera punctata Fabricius), maize billbug (Sphenophorusmaidis Chittenden)); flea beetles, cucumber beetles, rootworms, leafbeetles, potato beetles, and leafminers in the family Chrysomelidae(e.g., Colorado potato beetle (Leptinotarsa decemlineata Say), westerncorn rootworm (Diabrotica virgifera virgifera LeConte), northern cornrootworm (Diabrotica barberi Smith & Lawrence); southern corn rootworm(Diabrotica undecimpunctata howardi Barber), corn flea beetle(Chaetocnema pulicaria Melsheimer), crucifer flea beetle (Phyllotretacruciferae Goeze), grape colaspis (Colaspis brunnea Fabricius), cerealleaf beetle (Oulema melanopus Linnaeus), sunflower beetle (Zygogrammaexclamationis Fabricius)); beetles from the family Coccinellidae (e.g.Mexican bean beetle (Epilachna varivestis Mulsant); chafers and otherbeetles from the family Scarabaeidae (e.g., Japanese beetle (Popilliajaponica Newman), northern masked chafer (white grub) (Cyclocephalaborealis Arrow), southern masked chafer (white grub) (Cyclocephalaimmaculata Olivier), European chafer (Rhizotrogus majalis Razoumowsky),white grub (Phyllophaga crinita Burmeister), carrot beetle (Ligyrusgibbosus De Geer)); carpet beetles from the family Dermestidae;wireworms from the family Elateridae (e.g., Melanotus spp., Conoderusspp., Limonius spp., Agriotes spp., Ctenicera spp., Aeolus spp.); barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae (e.g. Eleodes spp). In addition it includes: adults andlarvae of the order Dermaptera including earwigs from the familyForficulidae (e.g., European earwig (Forficula auricularia Linnaeus),black earwig (Chelisoches morio Fabricius)); adults and nymphs of theorders Hemiptera and Homoptera such as, plant bugs from the familyMiridae, cicadas from the family Cicadidae, leafhoppers (e.g. Empoascaspp.) from the family Cicadellidae, planthoppers from the familiesFulgoroidea and Delphacidae, treehoppers from the family Membracidae,psyllids from the family Psyllidae, whiteflies from the familyAleyrodidae, aphids from the family Aphididae, phylloxera from thefamily Phylloxeridae, mealybugs from the family Pseudococcidae, scalesfrom the families Coccidae, Diaspididae and Margarodidae, lace bugs fromthe family Tingidae, stink bugs from the family Pentatomidae, cinch bugs(e.g., Blissus spp.) and other seed bugs from the family Lygaeidae,spittlebugs from the family Cercopidae squash bugs from the familyCoreidae, and red bugs and cotton stainers from the familyPyrrhocoridae.

Also included are adults and larvae of the order Acari (mites) such aswheat curl mite (Aceria tosichella Keifer), brown wheat mite (Petrobialatens Müller), spider mites and red mites in the family Tetranychidae(e.g., European red mite (Panonychus ulmi Koch), two spotted spider mite(Tetranychus urticae Koch), McDaniel mite (T. mcdanieli McGregor),carmine spider mite (T. cinnabarinus Boisduval), strawberry spider mite(T. turkestani Ugarov & Nikolski)), flat mites in the familyTenuipalpidae (e.g., citrus flat mite (Brevipalpus lewisi McGregor)),rust and bud mites in the family Eriophyidae and other foliar feedingmites and mites important in human and animal health, i.e. dust mites inthe family Epidermoptidae, follicle mites in the family Demodicidae,grain mites in the family Glycyphagidae, ticks in the order Ixodidae(e.g., deer tick (Ixodes scapularis Say), Australian paralysis tick(Ixodes holocyclus Neumann), American dog tick (Dermacentor variabilisSay), lone star tick (Amblyomma americanum Linnaeus) and scab and itchmites in the families Psoroptidae, Pyemotidae, and Sarcoptidae; adultsand immatures of the order Orthoptera including grasshoppers, locustsand crickets (e.g., migratory grasshoppers (e.g., Melanoplus sanguinipesFabricius (migratory grasshopper), M. differentialis Thomas(differential grasshopper), M. femurrubrum De Geer, (redleggedgrasshopper)), American grasshoppers (e.g., Schistocerca americanaDrury), desert locust (S. gregaria Forskal), migratory locust (Locustamigratoria Linnaeus), house cricket (Acheta domesticus Linnaeus), molecrickets (Gryllotalpa spp.)); adults and immatures of the order Dipteraincluding leafminers (e.g. Agromyza parvicornis Loew (corn blotchleafminer)), midges (e.g., Contarinia sorghicola Coquillett (sorghummidge), Mayetiola destructor Say (Hessian fly), Sitodiplosis mosellanaGéhin, (wheat midge), Neolasioptera murtfeldtiana Felt, (sunflower seedmidge)), fruit flies (Tephritidae), frit flies (e.g., Oscinella fritLinnaeus), maggots (e.g., Delia platura Meigen (seedcorn maggot) andother Delia spp., Meromyza americana Fitch (wheat stem maggot)), houseflies (e.g., Musca domestica Linnaeus), lesser house flies (e.g., Fanniacanicularis Linnaeus, F. femoralis Stein), stable flies (e.g., Stomoxyscalcitrans Linnaeus), face flies, horn flies, blow flies (e.g.,Chrysomya spp., Phormia spp.), and other muscoid fly pests, horse flies(e.g., Tabanus spp.), bot flies (e.g., Gastrophilus spp., Oestrus spp.),cattle grubs (e.g., Hypoderma spp.), deer flies (e.g., Chrysops spp.),keds (e.g., Melophagus ovinus Linnaeus) and other Brachycera, mosquitoes(e.g., Aedes spp., Anopheles spp., Culex spp.), black flies (e.g.,Prosimulium spp., Simulium spp.), biting midges, sand flies, sciarids,and other Nematocera; adults and immatures of the order Thysanopteraincluding onion thrips (Thrips tabaci Lindeman), grass thrips(Anaphothrips obscrurus Müller), tobacco thrips (Frankliniella fuscaHinds), western flower thrips (Frankliniella occidentalis Pergande),soybean thrips (Neohydatothrips variabilis Beach), citrus thrips(Scirthothrips citri Moulton) and other foliar feeding thrips; insectpests of the order Hymenoptera including sawflies (e.g. wheat stemsawfly (Cephus cinctus Norton)), ants (e.g., red carpenter ant(Camponotus ferrugineus Fabricius), black carpenter ant (C.pennsylvanicus De Geer), Pharaoh ant (Monomorium pharaonis Linnaeus),little fire ant (Wasmannia auropunctata Roger), fire ant (Solenopsisgeminata Fabricius), thief ant (Solenopsis molesta Say), red importedfire ant (S. invicta Buren), Argentine ant (Iridomyrmex humilis Mayr),crazy ant (Paratrechina longicornis Latreille), pavement ant(Tetramorium caespitum Linnaeus), cornfield ant (Lasius alienusFörster), odorous house ant (Tapinoma sessile Say)), bees (includingcarpenter bees), hornets, yellow jackets and wasps; insect pests of theorder Isoptera including the eastern subterranean termite(Reticulitermes flavipes Kollar), western subterranean termite (R.hesperus Banks), Formosan subterranean termite (Coptotermes formosanusShiraki), West Indian drywood termite (Incisitermes immigrans Snyder)and other termites of economic importance; insect pests of the orderThysanura such as silverfish (Lepisma saccharina Linnaeus) and firebrat(Thermobia domestica Packard); insect pests of the order Mallophaga andincluding the head louse (Pediculus humanus capitis De Geer), body louse(P. humanus humanus Linnaeus), chicken body louse (Menacanthusstramineus Nitzsch), dog biting louse (Trichodectes canis De Geer),fluff louse (Goniocotes gallinae De Geer), sheep body louse (Bovicolaovis Schrank), short-nosed cattle louse (Haematopinus eurysternusNitzsch), long-nosed cattle louse (Linognathus vituli Linnaeus) andother sucking and chewing parasitic lice that attack man and animals;insect pests of the order Siphonoptera including the oriental rat flea(Xenopsylla cheopis Rothschild), cat flea (Ctenocephalides felisBouche), dog flea (C. canis Curtis), hen flea (Ceratophyllus gallinaeSchrank), sticktight flea (Echidnophaga gallinacea Westwood), human flea(Pulex irritans Linnaeus) and other fleas afflicting mammals and birds.Additional arthropod pests covered include: spiders in the order Araneaesuch as the brown recluse spider (Loxosceles reclusa Gertsch & Mulaik)and the black widow spider (Latrodectus mactans Fabricius), andcentipedes in the order Scutigeromorpha such as the house centipede(Scutigera coleoptrata Linnaeus).

Compounds of the embodiments may show high activity against agronomicpests in the order Lepidoptera(e.g., Alabama argillacea Hübner (cottonleaf worm), Archips argyrospila Walker (fruit tree leaf roller), A.rosana Linnaeus (European leaf roller) and other Archips species, Chilosuppressalis Walker (rice stem borer), Cnaphalocrocis medinalis Guenée(rice leaf roller), Crambus caliginosellus Clemens (corn root webworm),C. teterrellus Zincken (bluegrass webworm), Diatraea grandiosella Dyar(southwestern corn borer), D. saccharalis Fabricius (surgarcane borer),Earias insulana Boisduval (spiny bollworm), E. vittella Fabricius(spotted bollworm), Helicoverpa armigera Hübner (American bollworm), H.zea Boddie (corn earworm or cotton bollworm), Heliothis virescensFabricius (tobacco budworm), Herpetogramma licarsisalis Walker (sodwebworm), Lobesia botrana Denis & Schiffermüller (European grape vinemoth), Pectinophora gossypiella Saunders (pink bollworm), Phyllocnistiscitrella Stainton (citrus leafminer), Pieris brassicae Linnaeus (largewhite butterfly), P. rapae Linnaeus (small white butterfly), Plutellaxylostella Linnaeus (diamondback moth), Spodoptera exigua Hübner (beetarmyworm), S. litura Fabricius (tobacco cutworm, cluster caterpillar),S. frugiperda J. E. Smith (fall armyworm), and Tuta absoluta Meyrick(tomato leafminer)).

Compounds of the embodiments may also have commercially significantactivity on agronomically important members from the order Homopteraincluding: Acyrthisiphon pisum Harris (pea aphid), Aphis craccivora Koch(cowpea aphid), A. fabae Scopoli (black bean aphid), A. gossypii Glover(cotton aphid, melon aphid), A. maidiradicis Forbes (corn root aphid),A. pomi De Geer (apple aphid), A. spiraecola Patch (spirea aphid),Aulacorthum solani Kaltenbach (foxglove aphid), Chaetosiphon fragaefoliiCockerell (strawberry aphid), Diuraphis noxia Kurdjumov/Mordvilko(Russian wheat aphid), Dysaphis plantaginea Paaserini (rosy appleaphid), Eriosoma lanigerum Hausmann (woolly apple aphid), Brevicorynebrassicae Linnaeus (cabbage aphid), Hyalopterus pruni Geoffroy (mealyplum aphid), Lipaphis erysimi Kaltenbach (turnip aphid), Metopolophiumdirrhodum Walker (cereal aphid), Macrosiphum euphorbiae Thomas (potatoaphid), Myzus persicae Sulzer (peach-potato aphid, green peach aphid),Nasonovia ribisnigri Mosley (lettuce aphid), Pemphigus spp. (root aphidsand gall aphids), Rhopalosiphum maidis Fitch (corn leaf aphid), R. padiLinnaeus (bird cherry-oat aphid), Schizaphis graminum Rondani(greenbug), Sipha flava Forbes, (yellow sugarcane aphid), Sitobionavenae Fabricius (English grain aphid), Therioaphis maculata Buckton(spotted alfalfa aphid), Toxoptera aurantii Boyer de Fonscolombe (blackcitrus aphid), and T. citricida Kirkaldy (brown citrus aphid); Adelgesspp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera);Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly), B.argentifolii Bellows & Perring (silverleaf whitefly), Dialeurodes citriAshmead (citrus whitefly), Trialeurodes abutiloneus (bandedwingedwhitefly) and T. vaporariorum Westwood (greenhouse whitefly); Empoascafabae Harris (potato leafhopper), Laodelphax striatellus Fallen (smallerbrown planthopper), Macrolestes quadrilineatus Forbes (asterleafhopper), Nephotettix cinticeps Uhler (green leafhopper), N.nigropictus Stål (rice leafhopper), Nilaparvata lugens Stål (brownplanthopper), Peregrinus maidis Ashmead (corn planthopper), Sogatellafurcifera Horvath (white-backed planthopper), Sogatodes orizicola Muir(rice delphacid), Typhlocyba pomaria McAtee white apple leafhopper,Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus(periodical cicada); Icerya purchasi Maskell (cottony cushion scale),Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citriRisso (citrus mealybug); Pseudococcus spp. (other mealybug complex);Cacopsylla pyricola Foerster (pear psylla), Trioza diospyri Ashmead(persimmon psylla).

Compounds of the embodiments may also have activity on members from theorder Hemiptera including: Acrosternum hilare Say (green stink bug),Anasa tristis De Geer (squash bug), Blissus leucopterus leucopterus Say(chinch bug), Corythuca gossypii Fabricius (cotton lace bug),Cyrtopeltis modesta Distant (tomato bug), Dysdercus suturellusHerrich-Schaffer (cotton stainer), Euschistus servus Say (brown stinkbug), Euschistus variolarius Palisot de Beauvois (one-spotted stinkbug), Graptostethus spp. (complex of seed bugs), Leptoglossus corculusSay (leaf-footed pine seed bug), Lygus lineolaris Palisot de Beauvois(tarnished plant bug), Nezara viridula Linnaeus (southern green stinkbug), Oebalus pugnax Fabricius (rice stink bug), Oncopeltus fasciatusDallas (large milkweed bug), Pseudatomoscelis seriatus Reuter (cottonfleahopper).

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

The following examples are provided by way of illustration, not by wayof limitation.

EXPERIMENTAL Example 1 Identification of Novel Cry8A/Cry8B Homologuesfrom Bt

A potentially novel Cry8A/Cry8B homologue was identified from Dupont Btstrains. A series of sequence analyses steps was then performed todetermine if the identified homologue was novel.

Sequencing Characterization of Potentially Novel Cry8A/Cry8B Homologue

Cloning of 88 kD Fragment of Cry8A/Cry8B Gene

PCR primers were designed for cloning an 88 kD fragment (including thetoxin domain) from the N-terminus of the potentially novel Cry8A/Cry8Bgene. The following PCR primers were used:

Cry 8AB-75576: GGATCCATGAGTCCAAATAATCAAAATG (SEQ ID NO: 3) Cry8AB-73694:GCAGTGAATGCCTTGTTTACGAATAC (SEQ ID NO: 4)

The PCR products were cloned into a TA vector. Constructs containing thepotentially novel Cry8A/Cry8B gene were then sequenced again.

Primary Sequence Analysis of Potential Novel Cry8A/Cry8B Gene

To assess the novelty of the selected sequence, the nucleic acidsequence data from the N-terminus and C-terminus (approximately 650 bpfrom each terminus) of the toxin domain for the potentially novelCry8A/Cry8B homologue was analyzed using BLAST searches against knownpesticidal genes from public Bt databases and published patents. Theentire 88 kD fragment for the potentially novel sequence was sequencedand cloned into an expression vector. Table 1 shows the percent identityof the toxin domain of the novel Cry8A/Cry8B gene and known Cry8 genes.

TABLE 1 Percent Identity of the Novel Cry8AB Homologue Toxin DomainCry8Aa Cry8Ba Cry8Bb Cry8Bc Cry8Ca Cry8Da Cry8AB008.1 (SEQ ID NO: 1)73.4 62.6 63.8 66.7 57.1 67.1Secondary Sequence Analysis of Potential Novel Cry8A/Cry8B Gene

The sequence data for the entire 88 kD fragment of the potentially novelCry8A/Cry8B homologue was analyzed using BLAST searches against knownpesticidal genes from public Bt databases and published patents. Thepercent identity of the 88 kD fragment relative to known pesticidalgenes was used to further assess the novelty of the selected sequence.

Final Sequence Analysis of Potentially Novel Cry8A/Cry8B Gene

Once the sequence was determined to be novel by the secondary sequenceanalysis, further analysis was performed by Southern blot and dot blot.Gene libraries for those Bt strains that harbor potential novelCry8A/Cry8B genes were generated, and the full-length sequence for thepotentially novel gene was determined. Genome-walking experiments wereperformed to confirm the novelty of the identified sequence.

Expression of Novel Cry8A/Cry8B Gene and Bioassays for PesticidalActivity

The DNA fragment representing 88 kDa for the novel Cry8A/Cry8B gene wascloned into pET20b expression vectors (Clontech). The His-Tagpolypeptides encoded by the novel genes were purified using Talon MetalAffinity Resin (BD Bioscience Clontech) and used in bioassays forassessing pesticidal activity against western corn rootworm (WCRW),Colorado potato beetle (CPB), and southern corn rootworm (SCRW). Suchbioassays are well known in the art. See, for example, Czapla and Lang(1990) J. Econ. Entomol. 83(6):2480-2485 and U.S. Pat. Nos. 6,570,005and 6,339,144. The results of the bioassay are summarized below in Table2.

TABLE 2 Pesticidal Activity of Novel Cry8A/Cry8B Toxins Pesticidal ToxinDomain Expression Activity Cry8AB008.1 Full Yes CPB active (SEQ ID NO:1)

Example 2 Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the novel Cry8A/Cry8B gene designated Cry8AB001.1(SEQ ID NO:1) operably linked to the ubiquitin promoter and theselectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37),which confers resistance to the herbicide Bialaphos. Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

A plasmid vector comprising the Cry8AB001.1 (SEQ ID NO:1) operablylinked to the ubiquitin promoter is made. This plasmid DNA plus plasmidDNA containing a PAT selectable marker is precipitated onto 1.1 μm(average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows: 100 μL prepared tungsten particles in water; 10 μL(1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μL 2.5 M CaC1₂; and10 μL 0.1 M spermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multi-tube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 mL 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μLspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/L Bialaphos,and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of Cry8AB001.1 by assaysknown in the art, such as, for example, immunoassays and westernblotting.

Analysis of Transgenic Maize Plants

Transgenic maize plants positive for expression of Cry8AB001.1 aretested for resistance to WCRW, CPB, and SCRW using standard bioassaysknown in the art. Such methods include, for example, root excisionbioassays and whole plant bioassays. See, e.g., U.S. Pat. PublicationNo. US 2003/0120054 and International Publication No. WO 03/018810.

Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMAC-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/Lthiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D, and 2.88 g/L L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/L Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/L silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/L N6 basalsalts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/L Gelrite (added after bringing to volume with D-I H₂O); and0.85 mg/L silver nitrate and 3.0 mg/L bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and 0.40 g/L glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/Lsucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/L Gelrite (addedafter bringing to volume with D-I H₂O); and 1.0 mg/L indoleacetic acidand 3.0 mg/L bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinicacid, 0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and 0.40 g/Lglycine brought to volume with polished D-I H₂O), 0.1 g/L myo-inositol,and 40.0 g/L sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/L bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 3 Agrobacterium-Mediated Transformation of Maize

For Agrobacterium-mediated transformation of maize with the Cry8AB001.1nucleotide sequence (SEQ ID NO:1), the method of Zhao is employed (U.S.Pat. No. 5,981,840, and International Pat. Publication No. WO 98/32326;the contents of which are hereby incorporated by reference). Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacterium, where the bacteria are capable oftransferring the Cry8AB001.1 gene to at least one cell of at least oneof the immature embryos (step 1: the infection step). In this step theimmature embryos are immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). The immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). The immature embryos are culturedon solid medium with antibiotic, but without a selecting agent, forelimination of Agrobacterium and for a resting phase for the infectedcells. Next, inoculated embryos are cultured on medium containing aselective agent and growing transformed callus is recovered (step 4: theselection step). The immature embryos are cultured on solid medium witha selective agent resulting in the selective growth of transformedcells. The callus is then regenerated into plants (step 5: theregeneration step), and calli grown on selective medium are cultured onsolid medium to regenerate the plants.

Example 4 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing the novelCry8A/Cry8B gene designated Cry8AB001.1 (SEQ ID NO:1) operably linked toa ubiquitin promoter as follows. To induce somatic embryos, cotyledons,3-5 mm in length dissected from surface-sterilized, immature seeds ofthe soybean cultivar A2872, are cultured in the light or dark at 26° C.on an appropriate agar medium for six to ten weeks. Somatic embryosproducing secondary embryos are then excised and placed into a suitableliquid medium. After repeated selection for clusters of somatic embryosthat multiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A Dupont Biolistic PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the Cry8AB001.1 (SEQ IDNO:1) operably linked to the ubiquitin promoter can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which theembodiments of this invention pertain. All publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: a) the nucleotide sequence setforth in SEQ ID NO:1; b) a nucleotide sequence encoding the amino acidsequence of SEQ ID NO:2; and c) a nucleotide sequence encoding apolypeptide having at least 95% sequence identity to SEQ ID NO:2,wherein said polypeptide has pesticidal activity.
 2. A DNA constructcomprising a nucleotide sequence of claim 1 operably linked to apromoter that drives expression in a plant.
 3. A transformed plant cellcomprising at least one polynucleotide construct that comprises aheterologous nucleotide sequence operably linked to a promoter thatdrives expression in the plant cell, wherein said nucleotide sequence isselected from the group consisting of: a) the nucleotide sequence setforth in SEQ ID NO: 1; b) a nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 2; and c) a nucleotide sequence encoding apolypeptide having at least 95% sequence identity to SEQ ID NO:2,wherein said polypeptide has pesticidal activity.
 4. A plant comprisingat least one polynucleotide construct that comprises a heterologousnucleotide sequence operably linked to a promoter that drives expressionin the plant, wherein said nucleotide sequence is selected from thegroup consisting of: a) the nucleotide sequence set forth in SEQ ID NO:1; b) a nucleotide sequence encoding the amino acid sequence of SEQ IDNO: 2; and c) a nucleotide sequence encoding a polypeptide having atleast 95% sequence identity to SEQ ID NO:2, wherein said polypeptide haspesticidal activity.
 5. The plant of claim 4, wherein saidpolynucleotide construct is stably incorporated into the genome of theplant.
 6. The plant of claim 5, wherein said plant displays increasedresistance to an insect pest.
 7. The plant of claim 6, wherein saidinsect pest is a Coleopteran pest.
 8. The plant of claim 6, wherein saidinsect pest is Colorado potato beetle.
 9. The plant of claim 4, whereinsaid promoter is a pathogen-inducible promoter.
 10. The plant of claim4, wherein said plant is a monocot.
 11. The plant of claim 4, whereinsaid plant is a dicot.
 12. The plant of claim 11, wherein said dicot ispotato, soybean, Brassica, sunflower, cotton, or alfalfa.
 13. Atransgenic seed of the plant of claim 10, wherein the seed comprisessaid polynucleotide construct.
 14. A method for protecting a plant froman insect pest, said method comprising introducing into said plant atleast one polynucleotide construct that comprises a heterologousnucleotide sequence operably linked to a promoter that drives expressionin the plant, wherein said nucleotide sequence is selected from thegroup consisting of: a) the nucleotide sequence set forth in SEQ IDNO:1; b) a nucleotide sequence encoding the amino acid sequence of SEQID NO: 2; and c) a nucleotide sequence encoding a polypeptide having atleast 95% sequence identity to SEQ ID NO:2, wherein said polypeptide haspesticidal activity.
 15. The method of claim 14, wherein said insectpest is a Coleopteran pest.
 16. The method of claim 14, wherein saidinsect pest is Colorado potato beetle (CPB).
 17. The method of claim 14,wherein said promoter is a pathogen-inducible promoter.
 18. The methodof claim 14, wherein said plant is a monocot.
 19. The method of claim14, wherein said plant is a dicot.
 20. The method of claim 19, whereinsaid dicot is potato, soybean, Brassica, sunflower, cotton, or alfalfa.